Expression of pneumococcal surface protein a (pspa)

ABSTRACT

The present invention relates to expression of Pneumococcal Surface Protein A (PspA). The invention represents an advancement in the field of genetic engineering and vaccine technology. The invention discloses expression vectors and recombinant host cells for expression of truncated PspA peptide. The invention also discloses vaccine compositions comprising the truncated peptides as carrier protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No. 16/976,794, filed on Aug. 31, 2020, which is a 371 U.S. National Stage Application of International Application No. PCT/IB2019/051655, filed on Mar. 1, 2019, which claims the benefit of IN Application No. 201841007814, filed on Mar. 1, 2018, the disclosures of which applications are incorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

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FIELD OF INVENTION

The present invention relates to high level expression of truncated Pneumococcal Surface Protein A1 (PspA1) in bacteria.

BACKGROUND

Streptococcus pneumoniae is an important cause of otitis media, meningitis, bacteremia and pneumonia, and a leading cause of fatal infections in the elderly and persons with underlying medical conditions. An attractive goal for streptococcal vaccination is to reduce carriage in the vaccinated populations and subsequently reduce the incidence of pneumococcal disease.

Pneumococcal polysaccharide vaccines marketed under the brand name Pneumovax23 is not effective in children under 2 years of age. The inefficacy of polysaccharide vaccines in this population has been attributed to the immaturity of the infant immune system in the expression of B cell receptors. Conjugation of polysaccharides (PS) to carrier proteins converts it from a T cell-independent antigen to a T cell-dependent antigen. As a T cell-dependent antigen, polysaccharides can raise a response with isotype switching, generation of memory cells and a boostable immunological response.

Membrane proteins exist within and span the membrane across which they serve to transport molecules or facilitate cell adhesion. The proteins may assist in the movement of substances by facilitated diffusion (i.e., passive transport) or active transport. The pneumococcal surface protein A (PspA) is a membrane protein and is another important virulence factor found attached to the cell wall of all Streptococcus pneumoniae strains.

The replacement of the universal carrier proteins, such as tetanus toxoid (TT) or CRM197, by a pneumococcal protein in particular PspA1 or a fragment thereof, besides broadening the vaccine coverage, would also prevent the impairment of immune responses caused by the excessive use of the same carrier proteins in conjugate vaccines. An expression plasmid is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is efficient production of protein, and this may be achieved by synthesizing significant amount of stable messenger RNA. It is possible to design an expression vector that exert a tight control of the expression, and the protein is only produced in high quantity when necessary through the use of suitable expression conditions. In absence of the tight control of the gene expression, the protein may also be expressed constitutively.

Corynebacterium glutamicum is a gram positive fermentative bacterium that is widely used in the production of mono-sodium glutamate, in high amounts. Owing to its stable genetic characteristics and lack of any endotoxin, Corynebacterium glutamicum is classified as GRAS organism (generally regarded safe). The bacterium is not known to secrete any extracellular proteases, hence it becomes an attractive platform for producing heterologous proteins into the medium. This can be achieved using an expression plasmid construct that can synthesize the recombinant protein in high amounts.

EP2310502 B1 discloses the use of P_(tac) promoter in a construct wherein an Escherichia coli strain containing IPTG inducible ftsZ and minCDE deletion mutation were grown in LB medium.

Nokano et. al., J Bacteriol. 1984 January; 157(1):79-83 discloses use of kanamycin resistance gene in the plasmid construct in order to make the transformed strain which acquire the property of being resistant to kanamycin.

Masai et. al., Proc Natl Acad Sci USA. 1987 July; 84(14):4781-5 discloses RepA protein for initiation of R1 plasmid replication and interact with oriR sequence.

Nayak et. al., Infect. Immun.-1998-Nayak-3744-51 discloses a live oral recombinant Salmonella vaccine strain expressing pneumococcal surface protein A (PspA).

Nabors et. al., Vaccine 18 (2000) 1743-54 discloses the expression of recombinant truncated PspA as cytoplasmic protein in Escherichia coli.

Figueredo et. al., Appl Microbiol Biotechnol (2017) 101:2305-2317 discloses Production and purification of an untagged recombinant pneumococcal surface protein A (PspA4Pro).

The above-mentioned references either disclose the genetic elements of the expression vector or expression of pneumococcal surface protein A in Escherichia coli and Salmonella.

The inventors have identified immunogenic fragments of Pneumococcal Surface Protein. Thereafter, the inventors have made extensive efforts to develop expression constructs capable of stable and constitutive or inducible expression of the truncated pneumococcal surface protein A1 (PspA1) in bacteria at high level.

Therefore, the present invention contemplates to overcome the challenges of the prior art by preparing expression vectors and recombinant host cells for expression of truncated pneumococcal surface proteins. Further, inventors have prepared vaccine compositions comprising the truncated proteins as carrier proteins.

Objective

The main objective of the present invention is to provide an expression construct for the high-level expression of truncated pneumococcal surface protein A1 (PspA1) in bacteria.

Another objective of the present invention is to provide an expression construct capable of stably and constitutively or inducibly expressing a high level of truncated pneumococcal surface protein A1 (PspA1) in bacteria.

SUMMARY

The present invention provides an expression construct capable of high level expression of truncated pneumococcal surface protein A1 (PspA1) as set forth in SEQ ID NO: 3 and 4.

The present invention provides an expression construct comprising a gene, which encodes for truncated pspA1, as set forth in SEQ ID NO: 5 and 6.

The present invention provides an expression construct for the high-level expression of truncated PspA1 (Pneumococcal Surface Protein A1) as set forth in SEQ ID NO: 3 or 4, in bacteria comprising:

-   -   a) gene encoding truncated pspA1 as set forth in SEQ ID NO: 5 or         6,     -   b) origin of replication,     -   c) Antibiotic resistance gene,     -   d) A promoter and     -   e) ribosomal binding site.

The present invention is also directed to a method for high level expression of truncated PspA1 (Pneumococcal Surface Protein A1) which comprises culturing of bacteria transformed with an expression construct and thereby purifying the expressed protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Panel A shows the diagrammatic representation of pBE31C. Panel B shows sschematic presentation of the domains of pspA delineated from the deduced amino acid sequence of Rx1 pspA1. Panel C shows the diagrammatic representation of pBE117. Panel D shows the PCR amplicon containing RBS, native signal peptide, truncated pspA 1, native terminator and trrnB.

FIG. 2 (SEQ ID NO: 28): Protein sequence coverage of truncated PspA1 in a peptide fingerprint analysis.

FIG. 3 : Intact mass analysis of the truncated PspA1 expressed in Corynebacterium glutamicum.

FIG. 4 : Truncated PspA1 eluted from ceramic hydroxyapatite (CHT-II) column.

FIG. 5 : Truncated PspA1 eluted from anion exchange column.

FIG. 6 : Truncated PspA1 after diafiltration.

FIG. 7 : SEC-HPLC chromatogram for conjugation reaction kinetics of pneumococcal polysaccharide Serotype 3 (A), 6A (B) and 6B (C).

FIG. 8 : Serum antibody titer of immunized rabbits against different conjugates of Streptococcus pneumoniae polysaccharide from serotypes 3, 6A, 6B (2.2 mcg) with truncated pspA1 carrier protein.

FIG. 9 : Serum antibody titer of immunized rabbits against different conjugates of Streptococcus pneumoniae polysaccharide from serotypes 3, 6A, 6B (4.4 mcg) with truncated pspA1 carrier protein.

FIG. 10 : Panel A shows the diagrammatic representation of pBE114k. Panel B shows the confirmation of pBE114k by restriction digest.

FIG. 11 : Truncated PspA1 eluted from CHT I chromatography.

FIG. 12 : Truncated PspA1 eluted from Capto Q Impress chromatography.

FIG. 13 (SEQ ID NO: 29): Protein coverage of PspA1 by MALDI.

FIG. 14 : Intact mass analysis of the truncated PspA1 expressed in Escherichia coli.

DETAILED DESCRIPTION

The term PspA1 refers to Pneumococcal Surface Protein A1 from Streptococcus pneumonia.

The present invention relates to high level expression of truncated PspA1 (Pneumococcal Surface Protein A1) in bacteria. The bacteria suitable for high level expression of pspA1 is Corynebacterium glutamicum and Escherichia coli.

In an embodiment, the present invention relates to high level expression of truncated PspA1 (Pneumococcal Surface Protein A1) as set forth in SEQ ID NO: 3 in Corynebacterium glutamicum.

In another embodiment, the present invention relates to high level expression of truncated PspA1 (Pneumococcal Surface Protein A1) as set forth in SEQ ID NO: 4 in Escherichia coli.

The present invention provides an expression construct capable of high level expression of surface protein as set forth in SEQ ID NO: 3.

The present invention also provides an expression construct capable of high level expression of surface protein as set forth in SEQ ID NO: 4.

In another embodiment, the present invention relates to an expression construct for high level expression of truncated PspA1 (Pneumococcal Surface Protein A1) as set forth in SEQ ID NO: 3 in Corynebacterium glutamicum.

In yet another embodiment, the present invention relates to an expression construct for high level expression of truncated PspA1 (Pneumococcal Surface Protein A1) as set forth in SEQ ID NO: 4 in Escherichia coli.

The present invention relates to an expression construct which is used for the high-level expression of truncated PspA1 in Corynebacterium glutamicum comprising:

-   -   i. ori R origin of replication,     -   ii. Kanamycin resistance gene,     -   iii. P_(tac) promoter,     -   iv. Gene of interest coding for a truncated pspA1 (SEQ ID NO:         5).

The present invention also relates to an expression construct which is used for the high-level expression of truncated PspA1 in Escherichia coli comprising:

-   -   i. pUC origin of replication     -   ii. Kanamycin resistance gene     -   iii. PT7 promoter     -   iv. Gene of interest coding for a truncated pspA1 (SEQ ID NO:         6).

In an embodiment, the expression construct for high level expression of truncated pspA1 further comprises ribosomal binding site (RBS). RBS is included in the forward primer and a short stretch of DNA containing native terminators are included in the reverse primer used for further amplification of truncated pspA1. The RBS (Ribosomal Binding Site) is Triose phosphate isomerase in the expression construct for the expression of truncated PspA1 in Corynebacterium glutamicum.

In an embodiment of the present invention, the expression construct comprises PspA1 signal peptide coding region (native), truncated pspA1, P_(tac) promoter and Ribosomal binding site (RBS) of triose phosphate isomerase gene.

In a preferred embodiment, the present invention provides an expression construct for high level expression of truncated PspA1 (SEQ ID NO: 3), in Corynebacterium glutamicum which comprises:

-   -   a. gene encoding truncated pspA1 (SEQ ID NO: 5),     -   b. ori R origin of replication (SEQ ID NO: 12),     -   c. kanamycin resistance gene (SEQ ID NO: 1),     -   d. P_(tac) promoter (SEQ ID NO: 2) and     -   e. triose phosphate isomerase ribosomal binding site.

In another preferred embodiment, the present invention provides an expression construct for high level expression of truncated PspA1 (SEQ ID NO: 4), in Escherichia coli which comprises:

-   -   a) gene encoding truncated pspA1 (SEQ ID NO: 6),     -   b) pUC origin of replication,     -   c) kanamycin resistance gene (SEQ ID NO: 1),     -   d) PT7 promoter (SEQ ID NO: 11), and     -   e) ribosomal binding site.

The pneumococcal surface protein A (PspA) is a membrane protein and is an important virulence factor found attached to the cell wall of all Streptococcus pneumoniae strains and is a promising component as has been shown to be highly immunogenic.

In a particular embodiment of the present invention, truncated PspA1 is used as a carrier protein. Carrier proteins, employed in the conjugate vaccines, are preferably proteins that are non-toxic and non-reactogenic and obtainable in large amount and purity. A carrier protein can be conjugated to capsular polysaccharide isolated from pathogenic bacteria to enhance immunogenicity of the polysaccharide. Carrier proteins should be amenable to standard chemical conjugation procedures.

The truncated PspA1 in Corynebacterium glutamicum is secreted into the extracellular medium. The secretion into the extracellular medium aids in efficient purification.

The present invention relates to high level expression of truncated PspA1 in Corynebacterium glutamicum wherein the N-terminal region along with proline rich region of truncated pspA1 gene was amplified from 23F capsular serotype of Streptococcus pneumoniae, together with the upstream region.

In an embodiment of the present invention, the expression construct comprises truncated pspA1, P_(tac) promoter and Ribosomal binding site (RBS) of triose phosphate isomerase gene.

In an embodiment of the present invention, the expression construct comprises truncated pspA1, PT7 promoter and Ribosomal binding site (RBS).

The expression construct is electroporated into Corynebacterium glutamicum and selected on LB plates with kanamycin as selectable marker.

P_(tac) is a strong hybrid promoter composed of the −35 region of the trp promoter and the −10 region of the lacUV5 promoter/operator.

pspA sequences were obtained from GenBank and aligned from the protein database. Primers were designed to specifically amplify native signal peptide coding region, N-terminal region along with the proline rich region of pspA genes belonging to families 1 & 2. The required regions of pspA1 and pspA2 were amplified from the available Streptococcus pneumoniae clinical isolates. MHC peptide analysis showed that pspA1 is comparatively more immunogenic than pspA2.

Amino acid sequence encoding truncated PspA1 expressed and produced in Corynebacterium glutamicum is set forth in SEQ ID NO: 3 having an intact mass observed to be 35452 Daltons.

Amino acid sequence encoding truncated PspA1 expressed and produced in Escherichia coli is set forth in SEQ ID NO: 4 having an intact mass observed to be 41966 Daltons.

DNA sequence encoding truncated pspA1 expressed in Corynebacterium glutamicum and Escherichia coli are set forth in SEQ ID NO: 5 and SEQ ID NO 6 respectively.

Corynebacterium glutamicum (previously known as Micrococcus glutamicus) used for expressing the truncated pspA1 is a GRAS organism Gram-positive, rod-shaped bacteria. Corynebacterium glutamicum is a GRAS organism. The whole genome sequence of Corynebacterium glutamicum ATCC 13032 is available which can be grown to higher cell densities and is also genetically stable owing to the lack of a recombination repair system. It has limited restriction-modification system. It shows no autolysis and can maintain metabolic activity under growth-arrested conditions. It has low protease activity favoring recombinant protein production. Its plasticity of metabolism and strong secondary metabolism properties, capacity to utilize broad spectrum of carbon sources (pentoses, hexoses, and alternative carbon sources), stress-tolerance to carbon sources make it a promising host for heterologous protein production. These physiological properties make Corynebacterium glutamicum accessible to manipulation and cultivation in robust industrial conditions, thus making it a successful industrial workhorse. Heterologous expression of proteins like α-Amylase, endoglucanase, endoxylanase, GFP, Xylanse etc., was reported in Corynebacterium glutamicum.

In an embodiment of the present invention, the yield of truncated PspA1 is about 500 mg/L, about 400 mg/L, about 300 mg/L, about 250 mg/L, about 220 mg/L, about 200 mg/L, about 180 mg/L, about 160 mg/L, about 150 mg/L, about 120 mg/L, about 100 mg/L.

In yet another embodiment, the present invention provides a pneumococcal conjugate vaccine comprising at least one polysaccharides from Streptococcus pneumoniae serotypes selected from 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 19A, 20A, 20B, 22F, 23A, 23B, 23F, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, 39 and 45, conjugated with truncated pspA1 of the present invention or combination of truncated PspA1 and other carrier proteins such as CRM197, tetanus toxoid, pertussis toxoid; PsaA and the like.

In yet another embodiment, the present invention provides multivalent pneumococcal vaccine composition selected from 10 valent, 14 valent, 15 valent, 17 valent, 18 valent, 19 valent, 20 valent, 22 valent, 23 valent, 24 valent or 25 valent comprising polysaccharides from Streptococcus pneumoniae serotypes selected from 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 19A, 20A, 20B, 22F, 23A, 23B, 23F, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, 39 and 45, conjugated with truncated pspA1 of the present invention or combination of truncated PspA1 and other carrier proteins such as CRM197, tetanus toxoid, pertussis toxoid; psaA and the like.

In a preferred embodiment, the present invention provides a multivalent conjugate vaccine comprising at least three polysaccharides from Streptococcus pneumoniae serotypes 3, 6A and 6B conjugated to truncated PspA1 of the present invention.

In an embodiment of the present invention provides a conjugate vaccine comprising polysaccharides from Streptococcus pneumoniae serotypes 3, 6A and 6B conjugated to truncated PspA1 of the present invention and Streptococcus pneumoniae serotypes 1, 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM197.

The present invention provides formulations containing either 2.2 μg or 4.4 μg of each of the pneumococcal polysaccharides from serotypes 3, 6A and 6B each conjugated to truncated PspA1 of the present invention and about 2.2 μg each of pneumococcal polysaccharides from serotypes 1, 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F each conjugated to CRM197.

In another embodiment, the present invention provides a pneumococcal vaccine composition as a single 0.5 mL dose, the single dose comprising about 2.2 to 4.4 μg of one or more pneumococcal polysaccharides; about 1 μg to about 50 μg of truncated PspA1 of the present invention conjugated to each of the one or more pneumococcal polysaccharides; about 0.2 mg to about 1 mg of aluminum phosphate adjuvant; and an excipient.

In another embodiment, the present invention provides a pneumococcal vaccine composition as a single 0.5 mL dose, the single dose comprising about 2.2 to 4.4 μg of one or more pneumococcal polysaccharides; about 1 μg to about 30 μg of truncated PspA1 of the present invention conjugated to each of the one or more pneumococcal polysaccharides; about 1 μg to about 30 μg of CRM197 conjugated to each of the one or more pneumococcal polysaccharides; about 0.2 mg to about 1 mg of aluminum phosphate adjuvant; and an excipient.

In another embodiment, the present invention also provides a vaccine for the prevention of invasive disease caused by Streptococcus pneumoniae, by administering the conjugated vaccine prepared by conjugating the truncated PspA1 of the present invention with pneumococcal polysaccharides.

EXAMPLES

The following examples are provided to illustrate the invention and are merely for illustrative purpose only and should not be construed to limit the scope of the invention.

Example 1: Recombinant Expression of Truncated PspA1 in Corynebacterium glutamicum

Construction of pBE31C

A small stably replicating broad host range plasmid pBE30 was generated using synthetic plasmid having 2.692 Kb pNG2 oriR sequence. Using this plasmid DNA as template, a 1.8 Kb oriR region was amplified using the primers pEP-F1 (5′-GCG CGG ACT AGT AGA TCT ATG GTA AAT CTG CGC AGA CAG-3′)(SEQ ID NO: 16) and pEP-R1 (5′-GCG CGG ACT AGT GAA TTC GGT GAG GTT ATG GCG-3′) (SEQ ID NO: 17).

Simultaneously, 1.033 Kb kanR sequence was amplified using pUC4-KIXX template DNA and Kan-F2 (5′-AAG GTC CCG GGA TGG CGA TAG CTA GAC TGG GCG GT-3′) (SEQ ID NO: 18) and Kan-R2 (5′-AAG GTC CCG GGG GTT GGG CGT CGC TTG GTC GG-3′) (SEQ ID NO: 19) primers. Both kanR gene amplicon and oriR amplicon were blunt end ligated to create pBE30 vector. Further, a 0.851 Kb expression cassette containing a tandem of tac lac UV5 promoter, multiple cloning site, lacZα component and TrrnB terminator sequence was amplified using the P_(tac)-F1 (5′-GG AGC ACT AGT CTG AAA TGA GCT GTT GAC AAT TAA TC-3′) (SEQ ID NO: 20) and P_(tac)-R1 (5′-GG AGC ACT AGT TTT AAA CAT GAG CGG ATA CAT ATT TGA A-3′) (SEQ ID NO: 21) primers each appending SpeI restriction site. The template DNA used for the amplification of expression cassette is pMMB206 (ATCC 37808). Later the expression cassette was cloned into the unique SpeI site designed in the pBE30 plasmid. The plasmid thus obtained is designated as pBE31C (FIG. 1A; SEQ ID NO: 14).

Construction of pBE117

Truncated pspA1 gene (N terminal region along with proline rich region FIG. 1B) was amplified from 23F capsular serotype of Streptococcus pneumoniae, together with the upstream region. This was cloned in a TA vector (pTZ57R/T procured from Fermentas) using primers PSPAF1_FP (5′ATG AAT AAG AAA AAA ATG ATT TTA ACA AGT CTA GCC 3′) (SEQ ID NO: 22) and PSPAF1_RP (5′CGA GAG AGA TCT AAA TTA AAA TGT CAA ATG TTC TTA ACA TGC TTT AAT TTT TAT TTT GGT GC 3′) (SEQ ID NO: 23) and sequence verified. This was designated as pTZ-pspA1. Native terminator sequence was included in the reverse primer PSPAF1_RP. The clade defining regions were mapped in the obtained truncated pspA1 sequence to confirm that it belongs to family 1 of PspA proteins.

To express truncated pspA1 in Corynebacterium glutamicum, P_(tac) promoter (SEQ ID NO: 2) and Ribosomal binding site (RBS) of triose phosphate isomerase gene (SEQ ID NO: 7) belonging to Corynebacterium, were chosen. pspA1 gene along with the entire cassette including RBS, native signal peptide (SEQ ID NO: 8), truncated pspA1 gene, native terminator (SEQ ID NO: 10) was amplified using primers—SDTICGR0949_FP5 (5′GAG CGA TGG ATC CTA GAA AGG TGT GTT TCA CCC ATG AAT AAG AAA AA 3′) (SEQ ID NO: 24) and PSPA2_2 RP (5′ TCA AAT GTT CTT AAC ATG CTT TAA TTT TTA TGG TGC AGG AGC TGG TTG 3′) (SEQ ID NO: 25) and pTZ-pspA 1 as template. RBS was included in the forward primer SDTICGR0949_FP5. rrnB terminator (SEQ ID NO: 13) region was amplified from pBE31C available in-house and ligated to gene encoding for truncated pspA1 using Splicing by Overlap Extension (SOE) PCR. rrnB gene was amplified using TER FP2 (5′ ATG TTA AGA ACA TTT GAC ATT TTA ATT TCG GCA CTG GCC GTC GTT 3′) (SEQ ID NO: 26) and TER_RP3 (5′GCG ATA TGG ATC CCA TGA GCG GAT ACA 3′) (SEQ ID NO: 27). PSPA2_2 RP and TER FP2 were designed such that there is an overlap of 17 bases. Both the amplicons pspA1 and rrnB were added in the molar ratio of 1:1 and used as template for SOE PCR with the primers SDTICGR0949_FP5 (5′GAG CGA TGG ATC CTA GAA AGG TGT GTT TCA CCC ATG AAT AAG AAA AA 3′) (SEQ ID NO: 24) and TER_RP3 (5′GCG ATA TGG ATC CCA TGA GCG GAT ACA 3′) (SEQ ID NO: 27). Subsequently, the entire amplicon including RBS, native signal peptide (SEQ ID NO: 8), truncated pspA1 gene, native terminator (SEQ ID NO: 10), rrnB terminator was digested with the restriction enzyme appended in the forward (SDTICGR0949_FP5) and reverse primers (TER_RP3) and cloned in an expression vector pBE31C made for Corynebacterium. The resulted clone was designated as an expression construct pBE117 (FIG. 1C; SEQ ID NO: 15). The expression vector along-with truncated pspA1 hereinafter is referred to as expression construct. The orientation of the insert (FIG. 1D) was confirmed by PCR analysis. The sequence of truncated pspA1 gene along with its expression cassette was confirmed by DNA sequencing.

Expression of Truncated PspA1

The expression construct was electroporated into Corynebacterium glutamicum ATCC 13032 and selected on LB plates with kanamycin as selectable marker. Twenty recombinant Corynebacterium glutamicum colonies were picked and analysed by PCR. Five colonies were chosen for constitutive expression of truncated pspA1. The recombinant colonies along with Corynebacterium glutamicum ATCC 13032 (as negative control) were inoculated into 10 ml Terrific Broth with 25 μg/ml final concentration of kanamycin and incubated at 35° C. with shaking at 200 rpm. After 16 h, secondary inoculation was done in 10 ml of the same medium described above, such that the final OD is 0.1. The cultures were incubated at 35° C. with shaking at 200 rpm for 18-20 h. After 18 h, the culture supernatants were checked for the expression of truncated pspA1. 30 μl of the supernatant was loaded onto 12% SDS-PAGE and analysed for truncated pspA1 expression. A prominent band was seen around 45 kDa.

Western analysis using N terminal epitope specific pspA polyclonal antibody (SantaCruz) confirmed the expression of truncated pspA1. The expression analysis of recombinant clone 5 was scaled up to 500 ml and the expression of truncated pspA1 was confirmed at least 3 times. Truncated pspA1 was initially purified from shake flask experiments using CHT type 1 and Capto Q impress to nearly 99% purity. Later, after confirming the consistent expression of truncated PspA1 in C. glutamicum, the expression was scaled up to 1.5 L.

Purification and Validation of Truncated PspA1

The 800 ml of culture supernatant containing truncated PspA1 from 1.6 L (0.66 mg/ml), obtained from upstream was dialysed through 10 kDa and concentrated to 260 ml (1.92 mg/ml). 70 ml of this dialysed concentrate was subjected to purification using CHT type 1 and Capto Q impress. The final recovery of truncated pspA1 was 162 mg/L.

MALDI MS/MS analysis of the gel plug containing purified truncated PspA1 from SDS-PAGE gave a clear hit score of 233 with Pneumococcal surface protein A. 31% (FIG. 2 ) sequence coverage was shown with the PspA protein with NCBI protein id ABY67187.1. The intact molecular weight analysis showed the molecular weight of the truncated pspA1 expressed is 35.4 kDa. This mass matches with the theoretical molecular weight of truncated pspA1. The peak at 17725.4 is a peak of molecule having a charge of 2, hence in the m/z the peak appears at half the intact mass of the truncated pspA1. (FIG. 3 )

Example 2: Production of Truncated PspA1

Corynebacterium glutamicum ATCC 13032 harbouring the expression construct comprising truncated pspA1 (hereinafter referred as pspA1) gene was revived from a source bank in LB media. This was used to inoculate a fermenter vessel (5 L; CSTR). The parameters that were monitored during the production process were—pH, DO, Temperature (AT), Carbon source, Metabolites. Process automation technology (PAT) based feeding strategy was adopted for carrying out Short Fed-Batch of truncated PspA1 fermentation. There was no induction-based control of the product (truncated PspA1) as this was growth associated product. The harvest OD was approximately 90 (OD_(600nm)) at the time of harvest in a semi-synthetic media. The cells were harvested and washed with PBS, prior to cell disruption in a French Press.

Purification of Truncated PspA1

16.2 L spent medium, with a total protein concentration of 0.8 mg/mL, was collected from 20 L fermentation batch. 16.2 L spent media was concentrated to 2.6 L (3.6 mg/mL) by using 10 kDa 0.5 m² cassette followed by Dia filtration against 20 mM Potassium phosphate pH-6.8, cond 3.2 ms/cm. This 2.6 L was split into two lots i.e Lot 1 with 1.4 L and Lot 2 with 1.2 L and proceeded to further purification. 500 ml of CHT I resin was packed in HiScale 50/40 column. Resin was washed with sterile distilled water followed by equilibration with 8 column volumes (CV) of 20 mM Potassium Phosphate pH-6.8 (Buffer A). 1400 mL (3.6 mg/mL) of the spent media concentrate (Lot 1) was loaded onto the column and flow through was collected. Column was washed with 5 column volumes of Buffer A. PspA1 was eluted with Step gradient using 250 mM Potassium Phosphate pH-6.8 (Buffer B). The step gradient involved 5 CVs of Step-40% B, 5 CVs of Step-80% B and 3 CVs of 0.5M Potassium Phosphate buffer. The flow rate was maintained at 80 mL/min for the entire run. PspA1 was collected in fraction 1 of Step-80% B with a fraction volume of 1250 mL. (FIG. 4 )

Capto Q Impress was used as a second chromatography step in PspA1 purification. 250 ml of Capto Q Impress resin was packed in XK 50/20 column. Resin was washed with sterile distilled water followed by equilibration with 5 column volumes (CV) of 20 mM Potassium Phosphate and 100 mM NaCl, pH-6.8 (Buffer A). 1250 mL of CHT I fraction was diluted to 2300 mL with 20 mM Potassium Phosphate, pH-6.8, loaded onto the column and flow through was collected. Column was washed with 5 column volumes of Buffer A. PspA1 was eluted using 12 CVs of Buffer B (20 mM Potassium Phosphate with 1 M NaCl, pH-6.2) in a linear gradient of 0 to 40% B and a final step of 3 CVs of 100% B. Each fraction of 250 mL was collected. The flow rate was maintained at 40 mL/min. PspA1 was collected in linear gradient of 40% B with pooled fraction volume of 1250 mL. (FIG. 5 )

Capto Q fractions of 4, 5, 6, 7 and 8 were pooled, concentrated/diafiltered with 20 mM Potassium phosphate pH-6.8. The final recovery obtained was 100 mL of pspA1 with a concentration of 15.5 mg/mL of protein (Total PspA1 is 1550 mg) from Lot 1. Similar process was followed for Lot 2 and the final recovery obtained was 70 mL of pspA1 with a concentration of 16 mg/mL (Total PspA1 is 1120 mg) from Lot 2. The purified PspA1 from both Lot 1 and Lot 2 is 2670 mg from 16.2 L batch (Purified PspA1 yield of 164 mg/L). (FIG. 6 )

Example 3: Construction of pBE114K

The expression vector pRSET A (commercial vector from Invitrogen) was modified by removing ampicillin resistance gene using DraI restriction digestion and ligating to SmaI digested kanamycin coding gene (obtained from pUC4 KIXX). This modified vector was designated as pRSET-km. Truncated PspA1 was expressed in Escherichia coli under PT7 promoter (SEQ ID NO: 11) of pRSET-km. A short stretch of DNA containing native terminators was included in the reverse primer used for further amplification of pspA1. Subsequently, the entire truncated pspA1 gene along with its native terminator was amplified, digested with the restriction enzyme appended in the forward and reverse primers and cloned in pRSET-km vector. The resulted clone was designated as pBE114k (FIG. 10A). The expression vector along-with truncated PspA1 hereinafter is referred to as expression construct. The expression construct pBE114k was confirmed by restriction digestion (FIG. 10B). The sequence of truncated PspA1 gene along with its expression cassette was confirmed by DNA sequencing.

Expression of Truncated PspA1

pBE114k was transformed into Escherichia coli DH5α-T1R chemical competent cells (Procured from Invitrogen) and selected on LB plates with kanamycin as selectable marker. 40 recombinant Escherichia coli colonies were picked and analysed by PCR. All the 40 colonies were chosen for inducible expression of truncated PspA1. The recombinant colonies along with Escherichia coli (as negative control) were inoculated into 10 ml Terrific Broth with 25 μg/ml final concentration of kanamycin and incubated at 37° C. with shaking at 200 rpm. At mid log phase, 1 mM IPTG was added to induce the expression of PspA1 in Escherichia coli (pBE114k). The cultures were incubated at 30° C. with shaking at 200 rpm for 16h, after induction. After 16 h, the culture supernatants were checked for the expression of truncated PspA1. The cells were collected by centrifugation, lysed and loaded onto 12% SDS-PAGE and analysed for truncated PspA1 expression. A prominent band was seen around 65 kDa. Western analysis using N-terminal epitope specific PspA polyclonal antibody (SantaCruz) confirmed the expression of truncated PspA1. The expression analysis of recombinant clone 29 was confirmed for the expression of truncated PspA1 at least 3 times. Truncated PspA1 was initially purified from shake flask experiments using CHT type 1 and Capto Q impress.

Purification and Validation of Truncated PspA1

40 grams (wet weight) of cell pellet from Escherichia coli (pBE114k) was taken and re-suspended in 400 ml of 20 mM Potassium Phosphate buffer pH-6.8 containing 1 mg/ml Lysozyme and 1 mM PMSF. The cell suspension was lysed using high pressure homogenizer for 3 passes at 1000 psi. 400 mL of cell lysate with a total protein concentration of 4 mg/mL was diluted to 750 mL with 20 mM Potassium Phosphate buffer pH-6.8 and purified using CHT I resin followed by Capto Q Impress as a second chromatography.

250 mL of CHT I resin was packed in HiScale 50/20 column. Resin was washed with sterile distilled water followed by equilibration with 5 column volumes (CV) of Buffer A. 750 mL diluted cell lysate was loaded onto the column and flow through was collected. Column was washed with 5 column volumes of Buffer A. PspA1 was eluted with Step gradient using 250 mM Potassium Phosphate pH-6.8, cond-29.5 ms/cm (Buffer B). The step gradient involved 5 CVs of Step-50% of Buffer B, 5 CVs of Step-80% of Buffer B and Column was stripped with 3 CVs of 0.5M Potassium Phosphate buffer pH-6.8, Cond-48 ms/cm (Buffer C). The flow rate was maintained at 40 mL/min for the entire run. The PspA1 protein peak fractions were collected manually, Elution fractions pool of 5, 6 and 7 of Step-80% B with a final volume of 350 mL (FIG. 11 ).

Capto Q Impress was used as a second chromatography step in PspA1 purification. 60 ml of Capto Q Impress resin was packed in XK 26/20 column. Resin was washed with sterile distilled water followed by equilibration with 5 column volumes (CV) of 20 mM Potassium Phosphate and 100 mM NaCl, pH-6.8, con-13.6 ms/cm (Buffer A). 350 mL of CHT I fraction was diluted to 700 mL with 1 mM Potassium Phosphate, pH-6.8, loaded onto the column and flow through was collected. Column was washed with 5 column volumes of Buffer A. PspA1 was eluted using linear gradient 10 CVs of Buffer B (20 mM Potassium Phosphate with 1 M NaCl, pH-6.2, cond-89 ms/cm) in a linear gradient of 0 to 40% B fractions were collected manually, fraction 4 to 8, 10 and 11 were 60 mL and 9 and 12 were 100 mL fractions (FIG. 12 ). Final step of 3 CVs of 100% B, 180 mL collected as fraction 13. The flow rate was maintained at 10 mL/min. The PspA1 protein was collected fraction 9 in linear gradient of 40% B was 100 mL.

Capto Q fraction of 9 was collected, concentrated/diafiltered with 20 mM Potassium phosphate pH-6.8. The final recovery obtained was 100 mL of PspA1 with a concentration of 5 mg/mL of protein (Total PspA1 protein is 500 mg) from 400 mL cell lysate with a concentration of 4 mg/mL. Recovery % is 31.25.

MALDI MS/MS analysis of the gel plug containing purified truncated PspA1 from SDS-PAGE gave a clear hit score of 223 with Pneumococcal surface protein A. 39% (FIG. 13 ) sequence coverage was shown with the PspA protein with NCBI protein id WP_050210652.1.

The intact molecular weight analysis showed the molecular weight of the truncated PspA1 expressed is 41.96 kDa. Intact mass analysis of the truncated PspA1 expressed in Escherichia coli showed 41966 Da (41.9 kDa), matches with the theoretical molecular weight of truncated PspA1. The peak at 20982.7 Da is a peak of molecule having a charge of 2 hence in the m/z the peak appears at half the intact mass of the truncated pspA1. (FIG. 14 ).

Example 4: Conjugation of Individual Pneumococcal Polysaccharide to Carrier Protein to Form Polysaccharide-Truncated PspA1 Conjugates

Activation of Serotype 3 and Conjugation with Truncated pspA1

Approximately, 1:1 ratio of sized serotype 3 (6.0 mL of PS, concentration of 10 mg/mL) and CDAP (100 mg/mL in Acetonitrile (w/v)) was mixed in a glass vial and stirred for 1 min. pH of pneumococcal polysaccharide serotype 3 were adjusted to 9.25 with 0.2M Triethylamine and stirred for 3 min at room temperature (RT). Truncated PspA1 (4.0 mL of conc. 15.0 mg/mL) were added to the activated serotype 3 in the ratio of 1:1 (Truncated PspA1: Serotype 3).

pH of the reaction was adjusted to ˜9.05 with 0.2M Triethylamine and the reaction was continued under stirring for 5 hours at room temperature and finally the reaction was quenched by adding excess concentration of glycine.

The reaction mixture was diafiltered using 100 kDa MWCO membrane and purified by size-exclusion chromatography wherein solid line is for polysaccharides and dashed lines for truncated pspA1 and five-hour reaction is represented by dotted line in chromatogram A (FIG. 7 ). The fractions were analysed by SEC-MALLS anthrone method and fractions containing conjugates were pooled and sterile filtered with 0.2μ filters. From now this material is called monovalent conjugate bulk (Serotype 3-Truncated PspA1 conjugate).

Activation of Serotype 6A and Conjugation with Truncated PspA1

Approximately, 1:1 ratio of sized serotype 6A (6.0 mL of PS, concentration of 10 mg/mL) and CDAP (100 mg/mL in Acetonitrile (w/v)) was mixed in a glass vial and stirred for 1 min. pH of pneumococcal polysaccharide serotype 6A were adjusted to 9.25 with 0.2M Triethylamine and stirred for 3 min at room temperature (RT). Truncated PspA1 (4.0 mL of conc. 15.0 mg/mL) were added to the activated serotype 6A in the ratio of 1:1 (Truncated PspA1:Serotype 6A).

pH of the reaction was adjusted to −0.9.05 with 0.2M Triethylamine and the reaction was continued under stirring for 5 hours at room temperature and finally the reaction was quenched by adding excess concentration of glycine.

The reaction mixture was diafiltered using 100 kDa MWCO membrane and purified by size-exclusion chromatography wherein solid line is for polysaccharides and dashed lines for truncated PspA1 and five-hour reaction is represented by dotted line in chromatogram B (FIG. 7 ). The fractions were analysed by SEC-MALLS, anthrone method and fractions containing conjugates were pooled and sterile filtered with 0.2μ filters. From now this material is called monovalent conjugate bulk (Serotype 6A-Truncated PspA1 conjugate).

Activation of Serotype 6B and Conjugation with Truncated PspA1

Approximately, 1:1 ratio of sized serotype 6B (6.0 mL of PS, concentration of 10 mg/mL) and CDAP (100 mg/mL in Acetonitrile (w/v)) was mixed in a glass vial and stirred for 1 min. pH of pneumococcal polysaccharide serotype 6B were adjusted to 9.25 with 0.2M Triethylamine and stirred for 3 min at room temperature (RT). Truncated PspA1 (4.0 mL of conc. 15.0 mg/mL) were added to the activated serotype 6B in the ratio of 1:1 (Truncated PspA1:Serotype 6B).

pH of the reaction was adjusted to −0.9.05 with 0.2M Triethylamine and the reaction was continued under stirring for 5 hours at room temperature and finally the reaction was quenched by adding excess concentration of glycine.

The reaction mixture was diafiltered using 100 kDa MWCO membrane and purified by size-exclusion chromatography wherein solid line is for polysaccharides and dashed lines for truncated PspA1 and five-hour reaction is represented by dotted line in chromatogram C (FIG. 7 ). The fractions were analysed by SEC-MALLS, anthrone method and fractions containing conjugates were pooled and sterile filtered with 0.2μ filters. From now this material is called monovalent conjugate bulk (Serotype 6B-Truncated PspA1 conjugate).

Example 5: Immunogenicity Study of the Conjugate Vaccine

Two formulations containing either 2.2 μg or 4.4 μg of Serotypes 3, 6A and 6B each conjugated to truncated PspA1 was prepared containing 2.2 μg of serotypes 1, 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F each conjugated to CRM197. These conjugates were adsorbed onto Al-hydrogel.

Rabbits having a body weight of 1.5 to 2 kg were grouped in 7 animals each and were immunized with the above-mentioned conjugate formulations. Sera samples were analysed before and after immunization. Serum obtained from the immunized rabbits were analysed for the presence of polysaccharide specific antibody in an indirect ELISA.

The serum antibody titer in the immunized rabbits were evaluated by indirect ELISA. Micro-titer plates coated with specific polysaccharides were reacted with the serum antibody. Rabbit serum prior to immunization, post 1^(st) and 2^(nd) dose was used for analysis wherein Y axis indicates antibody titer, which is arrived at using inverse of maximum dilution that gave ELISA OD₄₅₀ above the cut-off. Serum antibody titer in pre-immune rabbits were below detection limit. Open bar indicates titer after first dose of vaccine administration while the black solid bar indicates antibody titer after second dose of the vaccine (FIGS. 8 & 9 ). There was a dose dependent increase in both truncated pspA1 conjugated serotype as well as CRM197 conjugated serotype titers. The titers of CRM197 conjugated polysaccharide was not inhibited by the presence of truncated PspA1 conjugates and vice-versa. This indicates that the truncated PspA1 could be used as an alternative carrier protein for polysaccharide protein conjugate vaccine. 

I/We claim:
 1. A pneumococcal conjugate vaccine comprising at least one capsular polysaccharide from Streptococcus pneumoniae serotypes selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 19A, 20A, 20B, 22F, 23A, 23B, 23F, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, 39 and 45, wherein the polysaccharide is conjugated with a truncated PspA1 having a sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or a combination of truncated PspA1 and at least one other carrier protein selected from group of carrier proteins comprising of CRM197, tetanus toxoid, pertussis toxoid, and PsaA.
 2. The pneumococcal conjugate vaccine of claim 1, wherein the at least one capsular polysaccharide from Streptococcus pneumoniae serotypes is selected from the group consisting of 3, 6A and 6B.
 3. The pneumococcal conjugate vaccine of claim 1, wherein the vaccine is a multivalent vaccine selected from 10 valent, 14 valent, 15 valent, 17 valent, 18 valent, 19 valent, 20 valent, 22 valent, 23 valent, 24 valent and 25 valent.
 4. The pneumococcal conjugate vaccine of claim 1, wherein the multivalent vaccine is a 15 valent composition comprising pneumococcal capsular polysaccharides selected from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F.
 5. The pneumococcal conjugate vaccine of claim 1 comprising capsular polysaccharides from at least three Streptococcus pneumoniae serotypes 3, 6A and 6B conjugated to truncated PspA1 as set forth in SEQ ID NO: 3 or SEQ ID NO: 4, and capsular polysaccharides from Streptococcus pneumoniae serotypes 1, 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM197.
 6. The pneumococcal conjugate vaccine of claim 5, wherein the composition comprises: 2.2 to 4.4 μg of each of the capsular polysaccharides from Streptococcus pneumoniae serotypes 3, 6A and 6B conjugated to truncated PspA1; and 2.2 μg of each of the capsular polysaccharides from Streptococcus pneumoniae serotypes 1, 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM197.
 7. The pneumococcal conjugate vaccine of claim 1 wherein the pneumococcal vaccine composition is a single 0.5 mL dose comprising: about 2.2 to 4.4 μg of one or more pneumococcal capsular polysaccharides; about 1 μg to about 30 μg of truncated PspA1, having a sequence of SEQ ID NO: 3 or SEQ ID NO: 4, conjugated to each of the one or more pneumococcal capsular polysaccharides; about 0.2 mg to about 1 mg of aluminum phosphate adjuvant; and an excipient.
 8. The pneumococcal conjugate vaccine of claim 7, wherein the composition further comprises about 1 μg to about 30 μg of CRM₁₉₇ conjugated to each of the one or more pneumococcal capsular polysaccharides.
 9. The pneumococcal conjugate vaccine of claim 1, wherein truncated PspA1 is a carrier protein in a conjugate vaccine.
 10. The pneumococcal conjugate vaccine of claim 1, wherein the pneumococcal capsular polysaccharide is conjugated to the truncated PspA1 by a method comprising: a. preparing the truncated PspA1; b. activating the capsular polysaccharide from one or more pneumococcal serotypes to obtain an activated polysaccharide; and c. conjugating the activated polysaccharide with the truncated PspA1 prepared in step (a) to obtain a pneumococcal capsular polysaccharide-carrier protein conjugate.
 11. The pneumococcal conjugate vaccine of claim 10, wherein the truncated PspA1 is reacted with the activated polysaccharide in a ratio of 1:1.
 12. A method of conjugating a pneumococcal capsular polysaccharide to a truncated PspA1 having a sequence of SEQ ID NO: 3 or SEQ ID NO: 4 comprising: a. preparing the truncated PspA1; b. activating the capsular polysaccharide from one or more pneumococcal serotypes to obtain an activated polysaccharide; and c. conjugating the activated polysaccharide with the truncated PspA1 prepared in step (a) to obtain a pneumococcal capsular polysaccharide-carrier protein conjugate.
 13. The method of claim 12, wherein the truncated PspA1 is reacted with the activated polysaccharide in a ratio of 1:1.
 14. The method of claim 12, wherein the pneumococcal capsular polysaccharide comprises one or more Streptococcus pneumoniae serotypes selected from the group consisting of 1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 15C, 16F, 17F, 18C, 19F, 19A, 20A, 20B, 22F, 23A, 23B, 23F, 24B, 24F, 31, 33F, 34, 35B, 35F, 38, 39 and
 45. 15. The method of claim 14, wherein the capsular polysaccharide comprises one or more Streptococcus pneumoniae serotypes selected from the group consisting of 3, 6A and 6B.
 16. The method of claim 14 wherein the capsular polysaccharide comprises a Streptococcus pneumoniae serotypes selected from the group consisting of 1, 4, 5, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F and 33F conjugated to CRM₁₉₇.
 17. The method of claim 16, wherein the composition further comprises about 1 μg to about 30 μg of CRM₁₉₇ conjugated to each of the one or more pneumococcal capsular polysaccharides.
 18. The method of claim 12, wherein truncated PspA1 is a carrier protein in a conjugate vaccine.
 19. The method of claim 12, wherein the capsular polysaccharide is further conjugated to at least one other carrier protein selected from group of carrier proteins comprising of CRM197, tetanus toxoid, pertussis toxoid, and PsaA.
 20. The method of claim 12, wherein the pneumococcal capsular polysaccharide-carrier protein conjugate is in a conjugate vaccine. 